Recently, there has been increased need for periodically measuring the glucose level in blood (i.e., blood glucose) to diagnose and prevent diabetes. The blood glucose may be easily measured by using a hand-held and portable measuring device. Specifically, individual patients may easily measure blood glucose by using a biosensor in a strip form. Such a biosensor for measuring blood glucose is based on a colorimetric method or an electrochemical method as working principles.
Among these, the electrochemical method is explained by following Reaction Formula I and primarily characterized by using an electron transfer mediator. Examples of the electron transfer mediator may include ferrocene, and ferrocene derivatives; quinone, and quinine derivatives; organic and inorganic materials containing transition metals (e.g., hexamine ruthenium, osmium-containing polymers, potassium ferricyanide, etc.); and electron transfer organic materials such as organic conductive salts and viologen.
[Reaction Formula 1]Glucose+GOx-FAD→Gluconic acid+GOx-FADH2  (1)GOx-FADH2+an electron transfer mediator (in an oxidized state)→GOx-FAD+an electron transfer mediator (in a reduced state)  (2)
(In Reaction Formula 1, GOx indicates glucose oxidase; and GOx-FAD and GOx-FADH2 respectively indicate an oxidized state and a reduced state of flavin adenine dinucleotide (FAD) which is an active site of glucose oxidase.)
As shown in Reaction Formula 1, glucose in blood is firstly oxidized into gluconic acid by catalysis of glucose oxidase (1). In this step, FAD, which is an active site of glucose oxidase, is reduced to FADH2. Then, the reduced FADH2 is oxidized to FAD through a redox reaction with an electron transfer mediator and renders the electron transfer mediator reduced (2). The obtained electron transfer mediator in a reduced state can spread to a surface of an electrode. Then, the blood glucose concentration is determined by measuring electric current generated when an oxidation potential of the electron transfer mediator in a reduced state is applied to a surface of a working electrode.
Sometimes, if there is a need for reducing influence of oxygen in a blood sample, glucose dehydrogenase such as GDH-FAD is used in replacement of glucose oxidase GOx-FAD. Even though the types of enzymes are varied, the overall reaction follows the process in Reaction Formula 1.
A biosensor employing the above-mentioned electrochemical method as working principles is referred to as an electrochemical biosensor. In contrast to a biosensor using the conventional colorimetric method, the electrochemical biosensor is advantageous in that: influence of oxygen can be reduced; and a sample can be used without separate pretreatment even in the case where the sample is turbid.
Generally, although the electrochemical biosensor is conveniently used to monitor and control the blood glucose level, accuracy of the sensor is largely affected by various interfering species such as uric acid, acetaminophene, and ascorbic acid susceptible to oxidation.
Further, as a factor of causing a severe error in measurement accuracy of the electrochemical biosensor, erythrocyte volume fraction (i.e., a volumetric ratio of red blood cells in the whole blood; hematocrit) serves a key role. For people who regularly measure their blood glucose level by using a disposable biosensor strip, a biosensor, which is largely affected by hematocrit level, may bring about misjudgment in the measured result, and therefore cause even danger to lives of users.
Thus, hematocrit measurement accuracy is very important, since accuracy of the electrochemical biosensor in sensing hematocrit directly affects measurement accuracy of the blood glucose concentration eventually.
Patent documents 1 and 2 disclose a method for separating red blood cells, or a method for applying, to a reagent layer, a layer which eliminates red blood cells.
Patent document 3 discloses a method for using a screen-printable sensitive layer which includes silica filler and has an integrated function of reagent/blood cell separation.
Patent document 4 discloses a correction method of mathematically treating, through a chemometirc method, results obtained by applying an applied potential twice (i.e., the double excitation potential).
Measurement accuracy may be increased by providing an electrode for directly measuring hematocrit through electrical conductivity or resistance separately from an electrode for measuring an enzymatic reaction to separately measure hematocrit, and by using these results to correct a glucose concentration obtained from the enzymatic reaction measuring electrode. It has been suggested, as a prior art, a method capable of measuring hematocrit through conductivity of a disposable sensor equipped with an auxiliary electrode and a working electrode mounted on a sample cell in a capillary tube type (see Patent document 5), and also there is an example of applying this method to a biosensor for blood glucose measurement (see Patent document 6).
The present invention is focused on manufacturing of a sensor for accurately measuring conductivity in mass production of the sensor. Blood electrical conductivity (G) is based on Equation (1) below.G=σA/L  (1)
In Equation (1), G indicates electrical conductivity expressed in Ω−1 unit; σ indicates a conductivity coefficient of blood expressed in Ω−1 cm−1 unit; A indicates an area of an electrode expressed in cm2 unit; and L indicates a distance between electrodes expressed in cm unit. Accordingly, a constant distance between electrodes and constant area of the electrode are important for accurately measuring conductivity.
However, the prior art technique never discloses how to control the distance between electrodes and the area of the conductivity measuring electrode for measuring hematocrit to be constant in a mass-production type biosensor.
In the electrochemical biosensor, electrodes are formed by a printing method in most cases. However, during printing electrodes, deviations in a distance between electrodes and an area of the electrode are likely to occur, since printing is not carried out as exactly as desired depending on constituent materials, and an inclined edge of the electrode tends to slightly flow down. Also, as the printed electrode is getting thicker, a reaction occurred at the inclined edge has a greater effect on overall measurement. There is another problem in that, when even a small error occurs during electrode printing, areas of a working electrode and an auxiliary electrode are greatly varied. Such non-uniformity in electrode areas tends to be much severer in an electrode for measuring conductivity directly using an alternating current than in an electrode for an enzymatic reaction, thereby leading to a decrease in accuracy and precision of blood glucose measurement using hematocrit as a correction factor.
Therefore, while studying an electrochemical biosensor with more improved hematocrit measurement accuracy, the present inventors have completed the present invention by finding that, in an electrochemical biosensor including a first electrode part for correcting a measured value of hematocrit and a second electrode part for measuring a blood glucose concentration, an insulation cover is made thinner than a working electrode and an auxiliary electrode, so that areas of a first working electrode and a first auxiliary electrode of the first electrode part exposed to a blood sample become equal and electrode areas are maintained constantly by the insulation cover even when a positioning error occurs during printing.
Patent document 1: JP 2000338076 A
Patent document 2: U.S. Pat. No. 5,658,444 A1
Patent document 3: U.S. Pat. No. 6,241,862 B1
Patent document 4: WO 01/57510 A2
Patent document 5: U.S. Pat. No. 4,301,412 A1
Patent document 6: US 20110139634 A1.